Diane E. Capen

Mosaic tumor vessels : Cellular basis and ultrastructure of focal regions lacking endothelial cell markers

Endothelial cells of blood vessels in tumors may be thin, fragile, and defective in barrier function. We found previously that the endothelium of vessels in human colon carcinoma xenografts in mice is a mosaic structure. Approximately 85% of tumor vessels have uniform CD31 and/or CD105 immunoreactivity, but the remainder have focal regions that lack these common endothelial markers. The present study assessed the ultrastructure of the vessel lining and the integrity of the basement membrane in these regions. Using immunolabeling and confocal microscopy, we identified blood vessels that lacked CD31 and CD105 immunoreactivity and then analyzed the ultrastructure of these vessels by transmission electron microscopy. Eleven percent of vessels in orthotopic tumors and 24% of vessels in ectopic tumors had defects in CD31 and CD105 staining measuring on average 10.8 microm (range, 1-41.2 microm). Ultrastructural studies identified endothelial cells at 92% of CD31- and CD105-negative sites in orthotopic tumors and 70% of the sites in ectopic tumors. Thus, most regions of tumor vessels that lack CD31 and CD105 immunoreactivity represent attenuated endothelial cells with abnormal expression of endothelial cell markers, but some are gaps between endothelial cells. More than 80% of the defects lacked immunoreactivity for multiple basement membrane proteins.

Macrophages Facilitate Electrical Conduction in the Heart

Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes. Conversely, macrophages render the resting membrane potential of cardiomyocytes more positive and, according to computational modeling, accelerate their repolarization. Photostimulation of channelrhodopsin-2-expressing macrophages improves atrioventricular conduction, whereas conditional deletion of connexin 43 in macrophages and congenital lack of macrophages delay atrioventricular conduction. In the Cd11bDTR mouse, macrophage ablation induces progressive atrioventricular block. These observations implicate macrophages in normal and aberrant cardiac conduction.

Renal Atp6ap2/(Pro)renin Receptor Is Required for Normal Vacuolar H+-ATPase Function but Not for the Renin-Angiotensin System

ATPase H+-transporting lysosomal accessory protein 2 (Atp6ap2), also known as the (pro)renin receptor, is a type 1 transmembrane protein and an accessory subunit of the vacuolar H+-ATPase (V-ATPase) that may also function within the renin-angiotensin system. However, the contribution of Atp6ap2 to renin-angiotensin-dependent functions remains unconfirmed. Using mice with an inducible conditional deletion of Atp6ap2 in mouse renal epithelial cells, we found that decreased V-ATPase expression and activity in the intercalated cells of the collecting duct impaired acid-base regulation by the kidney. In addition, these mice suffered from marked polyuria resistant to desmopressin administration. Immunoblotting revealed downregulation of the medullary Na+-K+-2Cl- cotransporter NKCC2 in these mice compared with wild-type mice, an effect accompanied by a hypotonic medullary interstitium and impaired countercurrent multiplication. This phenotype correlated with strong autophagic defects in epithelial cells of medullary tubules. Notably, cells with high accumulation of the autophagosomal substrate p62 displayed the strongest reduction of NKCC2 expression. Finally, nephron-specific Atp6ap2 depletion did not affect angiotensin II production, angiotensin II-dependent BP regulation, or sodium handling in the kidney. Taken together, our results show that nephron-specific deletion of Atp6ap2 does not affect the renin-angiotensin system but causes a combination of renal concentration defects and distal renal tubular acidosis as a result of impaired V-ATPase activity.

PDGF and Microvessel Wall Remodeling in Adult Lung: Imaging PDGF-Rβ and PDGF-BB Molecules in Progenitor Smooth Muscle Cells Developing in Pulmonary Hypertension

Smooth muscle cells are relatively rare cells in the microvessels of the normal adult lung but develop in high numbers in the clinical pulmonary hypertensions (PHs). Understanding this cellular response has profound implications for determining the pathogenesis of PH, and for the development of therapeutic strategies, yet little is known of the angiogenic molecules responsible. The authors have previously shown that interstitial fibroblasts, and intermediate cells, are the progenitors of smooth muscle cells developing in adult lung microvessels in an in vivo model of experimental PH. The present study evaluates PDGF-Rβ/PDGF-BB, an important angiogenic signaling pathway, using antibodies linked to protein A-gold (pA-AU) and quantitative high-resolution imaging techniques to detect expression by these cells. Each progenitor cell type in the control lung expressed PDGF-Rβ and PDGF-BB. In the hypertensive lung, PDGF-Rβ was highly expressed by fibroblasts developing as perivascular cells, the mean number of pA-AU labeled antigenic sites per cell profile, and their density (μm−2), increasing with time: in intermediate cells the mean number of sites per cell profile, although not their density (μm−2), also increased with time but less so than in the fibroblasts. In clear contrast to the RTK, constitutive expression levels of PDGF-BB were low in each progenitor cell type and remained restricted in the hypertensive lung.

PDGF and microvessel wall remodeling in adult rat lung: imaging PDGF-AA and PDGF-Rα molecules in progenitor smooth muscle cells developing in experimental pulmonary hypertension

Smooth muscle cells are mostly absent from the walls of microvessels in the adult lung but develop in large numbers as part of the pathology of human and experimental pulmonary hypertensions (PHs). We have previously shown, in an in vivo model of experimental PH, that mesenchymal (interstitial) fibroblasts and intermediate cells are the progenitors of these cells. Although smooth muscle cell development is a defining pathophysiological feature of human PH, little is known about the angiogenic signaling molecules responsible. Here, we report data for platelet-derived growth factor AA (PDGF-AA) and PDGF-Rα, two components of an important signaling pathway for fibroblast and myofibroblast proliferation and migration. Using antibodies linked to protein-A gold and high-resolution imaging techniques, we analyzed the expression of these molecules as smooth muscle cells developed from progenitor cell populations and in endothelial cells of the same microvessels. PDGF-AA was highly expressed by each cell type in control lung. As PH developed, the number of antigenic sites for PDGF-AA decreased with time. PDGF-Rα expression levels in the control lung were low, relative to the ligand, and fell in PH. These data show, for the first time, a marked phenotypic shift in expression levels of the PDGF-AA isoform and its receptor tyrosine kinase in the progenitor smooth muscle cells developing in the microvessels of the adult hypertensive lung.

VEGFR2+PDGFRβ+ circulating precursor cells participate in capillary restoration after hyperoxia acute lung injury (HALI)

The in vivo morphology and phenotype of circulating cells that spontaneously contribute to new vessel formation in adults remain unclear. Here, we use high‐resolution imaging and flow cytometry to characterize the morphology and phenotype of a distinct population of circulating mononuclear cells contributing to spontaneous new vessel formation after hyperoxia acute lung injury (HALI). We identify a subpopulation of myeloid (CD11b/Mac1+) haematopoietic cells co‐expressing vascular endothelial growth factor receptor 2 (VEGFR2) and platelet derived growth factor receptor beta (PDGFRβ). Moreover, we show that these CD11b+VEGFR2+PDGFRβ+ circulating precursor cells (CPCs) contribute structurally to the luminal surface of capillaries re‐forming 2 weeks post‐HALI. This indicates that these myeloid CPCs may function, at least transiently, as putative vascular precursors, and has important implications for capillary growth and repair in injury and in pathologies of the lung and other organs.

A protocol for phenotypic detection and characterization of vascular cells of different origins in a lung neovascularization model in rodents

The goal of many current studies of neovascularization is to define the phenotype of vascular cell populations of different origins and to determine how such cells promote assembly of vascular channel. Here, we describe a protocol to immunophenotype vascular cells by high-resolution imaging and by fluorescence-activated flow cytometry in an in vivo rodent model of pulmonary microvascular remodeling. Analysis of cells by this combined approach will characterize their phenotype, quantify their number and identify their role in the assembly of vascular channels.

A protocol for a lung neovascularization model in rodents

By providing insight into the cellular events of vascular injury and repair, experimental model systems seek to promote timely therapeutic strategies for human disease. The goal of many current studies of neovascularization is to identify cells critical to the process and their role in vascular channel assembly. We propose here a protocol to analyze, in an in vivo rodent model, vessel and capillary remodeling (reorganization and growth) in the injured lung. Sequential analyses of stages in the assembly of vascular structures, and of relevant cell types, provide further opportunities to study the molecular and cellular determinants of lung neovascularization.

Intercalated Cell Depletion and Vacuolar H+-ATPase Mistargeting in an AE1 R607H Knockin Model

Distal nephron acid secretion is mediated by highly specialized type A intercalated cells (A-ICs), which contain vacuolar H+-ATPase (V-type ATPase)-rich vesicles that fuse with the apical plasma membrane on demand. Intracellular bicarbonate generated by luminal H+ secretion is removed by the basolateral anion-exchanger AE1. Chronically reduced renal acid excretion in distal renal tubular acidosis (dRTA) may lead to nephrocalcinosis and renal failure. Studies in MDCK monolayers led to the proposal of a dominant-negative trafficking mechanism to explain AE1-associated dominant dRTA. To test this hypothesis in vivo, we generated an Ae1 R607H knockin mouse, which corresponds to the most common dominant dRTA mutation in human AE1, R589H. Compared with wild-type mice, heterozygous and homozygous R607H knockin mice displayed incomplete dRTA characterized by compensatory upregulation of the Na+/HCO3- cotransporter NBCn1. Red blood cell Ae1-mediated anion-exchange activity and surface polypeptide expression did not change. Mutant mice expressed far less Ae1 in A-ICs, but basolateral targeting of the mutant protein was preserved. Notably, mutant mice also exhibited reduced expression of V-type ATPase and compromised targeting of this proton pump to the plasma membrane upon acid challenge. Accumulation of p62- and ubiquitin-positive material in A-ICs of knockin mice suggested a defect in the degradative pathway, which may explain the observed loss of A-ICs. R607H knockin did not affect type B intercalated cells. We propose that reduced basolateral anion-exchange activity in A-ICs inhibits trafficking and regulation of V-type ATPase, compromising luminal H+ secretion and possibly lysosomal acidification.

A quantitative ultrastructural study of circulating (monocytic) cells interacting with endothelial cells in high oxygen-injured and spontaneously re-forming (FVB) mouse lung capillaries

The present study demonstrates the fine structure of blood-borne (monocytic) circulating cells (CCs), and their interaction with endothelial cells, in a mouse model of lung capillary injury and repair. Quantitative analysis highlights the diversity of CC profiles entering the lung, where they form contact and adhesion/fusion sites to endothelial plasmalemmal membranes, and to complexes of endothelial/basement membrane remnants, as capillary networks reorganize over time. Temporal patterns of CC influx and efflux in the lung, changing CC phenotypes, and the range of CC interactions with endothelium, underscore the potential for a complex angiogenic/immunogenic response, as capillary networks stabilize and undergo expansion and growth.